Kidney Week

Abstract: TH-PO488

Resolution of Two Intronic Noncanonical Splicing Variants Located in X-linked Alport COL4A5 Gene

Session Information

• Genetic Kidney Diseases: Genotypes and Phenotypes in Cases
  October 24, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1202 Genetic Diseases of the Kidneys: Non-Cystic

Authors

• Lim, Si Ting, National University of Singapore Yong Loo Lin School of Medicine, Singapore, Singapore

Si Ting Lim

• Koh, Chee Teck, National University of Singapore Yong Loo Lin School of Medicine, Singapore, Singapore

Chee Teck Koh, MD

• Loh, Alwin Hwai Liang, Singapore General Hospital Division of Pathology, Singapore, Singapore

Alwin Hwai Liang Loh, MBChB

• Kwek, Jia Liang, Singapore General Hospital, Singapore, Singapore

Jia Liang Kwek, MBBS

• Choo Chon Jun, Jason, Singapore General Hospital, Singapore, Singapore

Jason Choo Chon Jun, MBBS, FASN

• Mok, Irene Yanjia, Singapore General Hospital, Singapore, Singapore

Irene Yanjia Mok, MBBS

• Lim, Cynthia Ciwei, Singapore General Hospital, Singapore, Singapore

Cynthia Ciwei Lim, MBBS

• Chin, Hui-Lin L., National University of Singapore Yong Loo Lin School of Medicine, Singapore, Singapore

Hui-Lin L. Chin, MBBS

• Ng, Jun Li, National University of Singapore Yong Loo Lin School of Medicine, Singapore, Singapore

Jun Li Ng

• Yap, Hui Kim, National University of Singapore Yong Loo Lin School of Medicine, Singapore, Singapore

Hui Kim Yap, MD, FASN

• Ng, Kar Hui, National University of Singapore Yong Loo Lin School of Medicine, Singapore, Singapore

Kar Hui Ng, MBBS

• Zhang, Yaochun, National University of Singapore Yong Loo Lin School of Medicine, Singapore, Singapore

Yaochun Zhang, PhD

Introduction

Variants located at intronic non-canonical sites are challenging to interpret due to limited literature. We report two families carrying two different intronic variants at non-canonical splicing sites of the COL4A5 gene which causes X-linked Alport syndrome.

Case Description

Proband 1 first presented at age 4 with isolated persistent microhematuria with no extrarenal manifestations. She progressed to A3 albuminuria by age 20, but kidney function remained normal at age 41. Her mother developed kidney failure at 60. Her son had persistent microhematuria and proteinuria since age 2, progressing to chronic kidney disease (CKD) by age 9. Proband 2 first presented at age 3 with microhematuria and recurrent gross hematuria and persistent subnephrotic proteinuria. He later developed stage 3 CKD and bilateral kidney cysts by age 14. His 44-year-old mother has microhematuria, persistent proteinuria with FSGS, bilateral renal cysts, but normal renal function. His 28-year-old maternal uncle was diagnosed with Alport syndrome at age 15, progressing to kidney failure and underwent a transplant. He has bilateral sensorineural hearing loss needing hearing aids.
In both probands, anti-COL4A5 immunofluorescence staining patterns were abnormal but not characteristic of classic X-lined or autosomal recessive disease. Electron microscopy showed GBM thinning for one case, while the other displayed thin and thick basement membranes with lamina densa multilamination.
Whole exome sequencing identified variant c.1032+4A>G in proband 1 and c.1032+3_1032+6delAAGT in proband 2, both located at intron 18 of COL4A5. To assess their impact on pre-mRNA splicing, exons 17-19 with introns 17-18 from probands and healthy controls were subcloned and transfected into HEK293 cells. The wild type construct produced a 300 bp band, while the mutant plasmids generated a truncated transcript. Sanger sequencing confirmed a skipping of exon 18.

Discussion

Skipping of exon 18 results in truncation of a critical domain of the protein, hence, both variants can be rendered a “strong” criterion in PVS1 according to the revised ACMG variant interpretation guidelines. This suggested both variants are likely pathogenic.